Biodistribution of ¹²⁵I-labeled antibodies in mice bearing OHS xenografts.

<p>Tumor to normal tissue ratios 24 hours after injection of ¹²⁵I-labeled antibodies in female nude mice bearing OHS tumor xenografts. Four to five animals were used for each radioimmunoconjugate, which gave from four to seven tumors per group. Error bars correspond to standard error of the mean.</p

Internalization.

<p>Representative images of internalization of HH1 and Rituximab in Ramos cells after incubation with 10 μg/ml of HH1 or 20 μg/ml rituximab at 4°C or 37°C for 1 hour or 19 hours, respectively. HH1-DOTA bound to Alexa Fluor 488 is shown in green, Rituximab bound to Alexa Fluor 647 is shown in magenta and Hoechst 33342 bound to DNA in the cell nucleus is shown in blue. 20 to 40 cells were scanned for each treatment.</p

Biodistribution of ¹⁷⁷Lu-OI-3 variants in mice bearing OHS xenografts.

<p>Comparison of biodistribution of ¹⁷⁷Lu-labeled murine OI-3, chimeric IgG1 OI-3 (CHOI-3.1) and chimeric IgG3 OI-3 (CHOI-3.3) in nude mice with OHS osteosarcoma xenografts. The data are presented as percentage of injected dose per gram tissue at 24 (A) and 48 hours (B) after injection, with error bars corresponding to the standard error of the mean. Three to six mice were used in each group, giving four to eight tumors per time point.</p

Effects of increasing dosage of ¹⁷⁷Lu-HH1 on the number of white blood cells, red blood cells and platelets.

<p><b>A</b>. Mice without xenografts treated with saline or 50, 100, 150 or 250 MBq/kg ¹⁷⁷Lu-HH1 (experiments 1 and 2). Blood samples were taken every 3 to 4 weeks, N = 4–10. Error bars  =  Standard deviation. <b>B</b>. Mice with Ramos xenografts treated with saline or 400, 800 or 1000 MBq/kg ¹⁷⁷Lu-HH1 (experiment 3 and 4). Blood samples were taken before treatment, 1 month after treatment and at euthanasia.</p

CD146 expression in human osteosarcoma cell lines.

<p>A representative flow cytometry histogram that compare the binding of murine anti-CD146 OI-3 antibody to three different human osteosarcoma cell lines: OHS (A), Saos-2 (B) and KPDX (C). Control samples are unstained cells and cells stained only with the FITC-conjugated secondary antibody (ab). In addition, the binding of murine anti-CD37 antibody HH1 was examined for OHS (A) and Saos-2 (B) cells. The samples were analyzed on a BD FacsCalibur.</p

Survival of mice treated with increasing dosages of ¹⁷⁷Lu-HH1.

<p><b>A</b>. Mice without xenografts treated with saline or 50, 100, 150 or 250 MBq/kg ¹⁷⁷Lu-HH1 (experiments 1 and 2). The end point for survival was euthanasia due to weight loss or substantial discomfort. <b>B</b>. Mice with Ramos xenografts treated with saline or 400, 800 or 1000 MBq/kg ¹⁷⁷Lu-HH1 (experiment 4). The end point for survival was euthanasia due to WBC<1.5 10⁹ cells/L (usually accompanied by RBC<5 10¹² and PLT<400 10⁹ cells/L), weight loss higher than 15% from base line, and/or substantial discomfort, all symptoms associated with radiation toxicity. Mice euthanized because of tumor diameter larger than 20 mm were censored.</p

Comparison of the binding ability of chimeric antibodies targeting different antigens on OHS cells.

<p>A representative flow cytometry histogram which shows binding of the chimeric versions of OI-3 to OHS cells, compared with samples incubated with cetuximab, trastuzumab and rituximab. Control samples are unstained cells and cells stained only with the FITC-conjugated secondary antibody (ab). The samples were run on a Guava EasyCyte HT. Take note that the samples with chimeric antibodies were run on a different platform, with different settings and secondary antibody, and the fluorescence intensity levels are therefore not directly comparable to runs with murine OI-3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165382#pone.0165382.g001" target="_blank">Fig 1</a>.</p

Histology: summary of main findings.

<p>Histology: summary of main findings.</p

Absorbed radiation doses to normal tissues and tumors.

<p>Estimated absorbed radiation dose (Gy) to normal tissues and tumors for nude mice with OHS osteosarcoma xenografts after intravenous injection of ¹⁷⁷Lu-labeled chimeric OI-3 IgG1 isotype antibody (CHOI-3.1). The data were normalized to an injected activity of 1 MBq per mouse. Error bars correspond to standard deviation.</p

Overview of experiments.

<p>Overview of experiments.</p